Farmacogenetica: come sfruttare le conoscenze biologiche per ridurre le tossicità e incrementare il sinegismo terpeutico.

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1 Farmacogenetica: come sfruttare le conoscenze biologiche per ridurre le tossicità e incrementare il sinegismo terpeutico Dr Valentina Citi Dip. Medicina Clinica e Sperimentale Università di Pisa

2 Tipologie di marker molecolari Diagnostici Identificazione di patologie (cromosoma Philadelphia in CML) Prognostici Associazione con l outcome clinico (BRAF V600E tumore del colon) Predittivi Attività di terapie specifiche (HER-2 e trastuzumab)

3 Tipologie di marker molecolari PREDITTIVI Biomarker predittivi Drug activity Drug toxicity EGFR - lung ALK - lung BRAF - melanoma BCR-ABL - AML HER-2 - breast DPD 5-FU UGT irinotecan TPMT 6-MP CDA gemcitabine

4 Tipologie di marker molecolari PREDITTIVI Biomarker predittivi Drug activity Drug toxicity EGFR - lung ALK - lung BRAF - melanoma BCR-ABL - AML HER-2 - breast DPD 5-FU UGT irinotecan TPMT 6-MP CDA gemcitabine

5 Quesito

6 Fluoropirimidine Farmaci antimetaboliti analoghi delle basi pirimidiniche Potenti inibitori della timidilato sintetasi Ampiamente utilizzati per il trattamento di molti tumori solidi, tra cui tumore del colon, della mammella e testa-collo. 5-FU Capecitabine Tegafur

7 L inattivazione del 5-FU dipende principalmente dalla diidropirimidina deidrogenasi Normal Deficiency 5-FU 5-FU 5-FdUMP 5-FdUMP TS DPD TS Tolerable toxicity 5-FDHU 5-FDHU Severe toxicity Evitare tossicità mortale a seguito del trattamento con 5-FU è un aspetto di rilevanza clinica

8 Time Line: valutazione dell attività della DPD e della tossicità da 5-FU presso il nostro dipartimento Enzymatic activity of DPD in PBMC Test dose of 5-FU 250 mg/sqm i.v. and concentration measurements DPD genotyping Factors to be considered for the choice of methods: 1) Minimal assay variability 2) Easy procedure 3) Widespread availability 4) Affordable cost

9 DPD deficiency e 5-FU toxicity Esone Introne C>T 62G>A 74A>G 85T>C 100delA 257C>T delTCAT 496A>G 601A>C 632A>G 703C>T 812delT 1003G>T 1039delTG 1108A>G 1156G>T 1896T>C 1897delC IVS14+1G>A 1601G>A 1627A>G 1679T>G 1714C>G 2657G>A 2846A>T 2933A>G 2983G>T 1475 C>T 2194G>A Del Re M et al. EPMA Journal 2011

10 Clinical data: patient #1 Exon Intron T>C 496A>G 1601G>A 1627A>G DIARRHEA 4 NAUSEA/VOMITING 3 STOMATITIS 3 NEUTROPENIA 3 THROMBOCYTOPENIA G>C 1896T>C IVS14+1GA 2194G>A 1st cycle OXALIPLATIN 85 mg/m², FOLINIC ACID 200 mg/m² 5-FU i.v. bolus 400 mg/m² followed by a 44-h continuous 400 mg/m² i.v. infusion

11 DEATH Clinical data: patient #2

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13 Raccomandazioni cliniche La terapia con fluoropirimidine è controindicata nei pazienti con gene DPYD mutato in omozigosi per le varianti DPYD*2A, c.1679t>g e c.2846a>t, poiché esse annullano l attività enzimatica DPD, mentre è necessario ridurre il dosaggio della fluoropirimidina almeno del 50% nei pazienti portatori delle mutazioni DPYD*2A, c.1679t>g e c.2846a>t in eterozigosi. La modifica della dose dovrà, inoltre, considerare anche eventuali trattamenti concomitanti.

14 Risposta

15 Tipologie di marker molecolari PREDITTIVI Biomarker predittivi Drug activity Drug toxicity Response Resistance

16 Tipologie di marker molecolari PREDITTIVI Biomarker predittivi Drug activity Drug toxicity Response Resistance Evolving tumor

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18 Quesito

19 Due meccanismi, ma non mutualmente esclusivi Adaptation resistant cells Zong Y, Goldstein AS. Nat Rev Urol. 2013;10(2):90-8

20 Due meccanismi, ma non mutualmente esclusivi Adaptation Clonal selection resistant cells resistant cells Zong Y, Goldstein AS. Nat Rev Urol. 2013;10(2):90-8

21 Scientific background NSCLC is an heterogeneous disease with distinct molecular characteristics Specific activating mutations in the tyrosine kinase domain of EGFR or ALK translocations are associated with sensitivity to TKIs (N Engl J Med 2004; Eur J Cancer 2012) Metastatic tumors often carry different genetic clones. Therefore, at tumor progression, further analysis of molecular markers is warranted (Lung Cancer 2013) It is well known that treatment acquired resistance to TKIs is associated with the acquired of secondary EGFR mutation (i.e. p.t790m) or ALK point mutations (i.e. p.l1196m) (N Engl J Med 2005; J Clin Oncol 2013)

22 Mechanisms of EGFR-TKIs acquired resistance J Clin Oncol Nov 1;31(31):

23 Mechanisms of ALK-TKIs acquired resistance J Clin Oncol Nov 1;31(31):

24 What s the matter? Several factors limit the feasibility of a re-biopsy and molecular analysis: amount of material that can be recovered during bronchial endoscopy difficult access to some tumor sites invasive nature of sampling methods possible dissemination of tumor cells

25 Review Review M olecular analysis of cell-free circulating DNA for the diagnosis of somatic mutations associated with resistance to tyrosine kinase inhibitors in non-small-cell lung cancer Molecular analysis of cell-free circulating DNA for the diagnosis of somatic mutations associated with resistance to tyrosine kinase inhibitors in non-small-cell Expert Rev. Mol. Diagn. Early online, 1 16 (2014) lung cancer Del Re 1, In non-small-cell lung cancer, the molecular diagnosis of somatic mutations is instrumental for Marzia Del Re, Enrico Vasile, Alfredo Falcone, Romano Danesi and Iacopo Petrini Enrico Vasile 2, Expert the Rev. choice Mol. of Diagn. the Early most online, appropriate 1 16 (2014) treatment. However, despite an initial response, resistance Alfredo Falcone 2, to tyrosine kinase inhibitors occurs and thereafter tumors progress. For this reason, next Romano Danesi* 1 generation inhibitors able to overcome acquired resistances are currently in development. and Therefore, the identification of the molecular determinants of resistance is needed to adapt

26 DNA circolante è quantificabile nel plasma Viable cells Dead cells cftdna Blood Perkins G, et al. PLoS ONE 7(11): e47020, 2012

27 Aim of the study Acquired resistance determines tumor progression Metastatic tumors have different genetic clones One drug-therapy could not be suitable to treat an heterogenous tumor Daily monitoring of patients during pharmacological treatment for the detection of acquired mutations in cftdna.

28 Study design, patients and methods 44 NSCLC patients were included in this analysis 33 EGFR+ NSCLC patients All patients received gefitinib/erlotinib and underwent disease progression 11 ALK+ NSCLC patients All patients received crizotinib and underwent disease progression Blood samples were collected at tumor progression and cftdna was extracted by QIAamp circulating nucleic acid kit (Qiagen ) cftdna was analysed by the ddpcr (BioRad ) to evaluate the appearance of resistance acquired mutations (codon 12 KRAS, p.t790m EGFR, ALK point mutations)

29 Results EGFR+ patients o p.t790m was detected in 11 subjects (33.3%) alone and in 13 patients (39.4%) with mutant KRAS o KRAS mutation at codon 12 alone or in combination with p.t790m was demonstrated in 3 (9.1%) o Six patients (18.2%) were negative for both KRAS and p.t790m ALK+ patients o ALK point mutations (p.l1196m, p.g1269a) were detected in 2 subjects (18,2%) in combination with mutant KRAS o KRAS mutation at codon 12 alone was demonstrated in 8 (72,7%) patients o Three patients (27,2%) were negative for both KRAS and ALK point mutations

30 Sample ID 36 - EGFR p.t790m at gefitinib progression Mutant allele amplification Wild type allele amplification

31 Sample ID 36 - EGFR p.t790m in response to AZD9291 Mutant allele amplification Wild type allele amplification

32 Sample ID 42 - ALK p.l1196m at crizotinib progression Mutant allele amplification Wild type allele amplification

33 Sample ID 42 ALK p.l1196m in response to AP26113 Disappearance of mutant allele Wild type allele amplification

34 Risposta

35 Conclusioni Tossicità da fluoropirimidine Le varianti DPYD*2A, c.1679t>g, c.2846a>t potrebbero essere associate a gravi tossicità a seguito della somministrazione di fluoropirimidine L analisi di questi polimorfismi pre-trattamento potrebbe evitare eventi di tossicità grave Il costo dell analisi viene ammortizzata dal miglioramento dello stato di salute del paziente Resistenza farmacologica L analisi molecolare di biomarker predittivi di resistenza dovrebbe essere incorporata nella pratica clinica Incrementare l utilizzo di nuove tecnologie dovrebbe essere discusso tra clinici e farmacologi Il costo dell analisi molecolare viene abbattuto dal miglioramento dell outcome clinico del paziente

36 Grazie per l attenzione Lab staff: Prof. Romano Danesi Dr Marzia Del Re Dr Valentina Citi Dr Marta Palombi Dr Francesca Belcari Ringraziamenti Tiseo M, Bordi P, Ardizzoni A, D Incecco A, Cappuzzo F, Petrini I, Lucchesi M, Vasile E, Falcole A, Chella A Camerini A, Amoroso A Inno A, Gori S Spada D, Testa E Malpeli G, Scarpa A